Distribution and dynamics of transcription-associated proteins during parvovirus infection.

Canine parvovirus (CPV) infection leads to reorganization of nuclear proteinaceous subcompartments. Our studies showed that virus infection causes a time-dependent increase in the amount of viral nonstructural protein NS1 mRNA. Fluorescence recovery after photobleaching showed that the recovery kinetics of nuclear transcription-associated proteins, TATA binding protein (TBP), transcription factor IIB (TFIIB), and poly(A) binding protein nuclear 1 (PABPN1) were different in infected and noninfected cells, pointing to virus-induced alterations in binding dynamics of these proteins.

Protein diffusion in mammalian cell cytoplasm.

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.